The present invention relates to particular binding agents which can be used in the treatment of inflammatory, autoimmune or allergic diseases.
CD23 (FCxcex5RII) is a type II molecule of the C-lectin family which also includes the lymphocyte homing receptor (MEL-14) and the endothelial leukocyte adhesion molecule-1 (ELAM 1). It is a low affinity receptor for IgE. In humans a variety of haematopoietic cell types express CD23 on their surface, including follicular dendritic cells, B cells, T cells and macrophages. CD23 molecules are also found in soluble forms in biological fluids. Soluble CD23 (sCD23) molecules are formed by proteolytic cleavage of transmembrane receptors. CD23 has pleiotropic activities including mediation of cell adhesion, regulation of IgE and histamine release, rescue of B cells from apoptosis and regulation of myeloid cell growth. These functional activities are mediated through the binding to specific ligands of cell-associated CD23, or sCD23, the latter acting in a cytokine-like manner (Conrad, D. H., Annu Rev Immunol 8, 623-645 1990); Delespesse, G., et al., Adv Immunol 49, 149-191 (1991); Bonnefoy, J. Y., et al., Curr Opin Immunol 5,944-947 (1993)).
Increased expression of CD23 has been observed in a number of inflammatory diseases. CD23 has been identified in synovial biopsies from patients with chronic synovitis, and sCD23 can be measured at concentrations exceeding the normal range in the serum and synovial fluid of patients with rheumatoid arthritis (Bansal, A. S., Oliver, W., Marsh, M. N., Pumphrey, R. S., and Wilson, P. B., Immunology 79, 285289 (1993); Hellen, E. A., Rowlands, D. C., Hansel, T. T., Kitas, G. D., and Crocker, J. J., Clin Pathol 44, 293-296 (1991); Chomarat, P., Brioloay, J., Banchereau, J., and Miossec, P., Arthritis Rheum 86, 234-242 (1993); Bansal, A., et al, Clin Exp Immunol 89, 452-455 (1992); Rezonzew, R., and Newkirk, M. M., Clin Immunol Immunopathol 71, 156-163 (1994)). In addition, levels of serum sCD23 in rheumatoid arthritis patients are related to disease status and correlate with serum rheumatoid factor (Bansal, A. S., et al., Clin Exp Rheumatol 12, 281-285 (1994)). Pro-inflammatory cytokines appear to be particularly important in rheumatoid arthritis, and a central role for TNF-xcex1 and IL-1xcex2 in the destruction of arthritic joints has been postulated (Brennan, F. M., Chantry, D., Jackson, A., Maini, R., and Feldman, M., Lancet 2, 244-247 (1989); Brennan, F. M., Maini, R. M., and Feldman M., Br J Rheumatol 31, 293-298 (1992)).
It has also been postulated that CD23-CD21 interactions may play a role in the control of IgE production (Flores-Romo L. et al., Science 261 1038-1041 (1993); Aubry et al., Nature 358, 505-507 (1992)).
CD11b and CD11c are adhesion molecules Fat participate in many cell-cell and cell-matrix interactions. CD11b/CD18 and CD11/CD18 (an association of CD11b and CD18 and of CD11c and CD18 respectively) have been reported to bind several ligands, including CD54, fibrinogen, factor X, LPS, Con A and zymosan (Springer, T. A., Nature 346, 425-434(1990)). The role of these binding molecules is not however completely understood. CD11b/CD18 and CD11c/CD18 are also known as MAC-1 and p150, 95 respectively. They are members of the xcex22 integrin family (sometimes known as Leu-CAM, ie leucocyte cell adhesion molecules). This family also includes LFA-1 amongst its members (also known as CD11a/CD18).
EP 0205405 purports to disclose Mabs to lymphocyte cellular receptors for IgE (FCxcex5R) cross reacting with human immunoglobulin E binding factor (IgE-BF), and derivatives thereof.
WO 93/04173 purports to disclose a polypeptide which is capable of binding to one of FCEL (low affinity IgE receptor FCxcex5RII) or FCEH (high affinity receptor FCxcex5RI) but which is substantially incapable of binding to the other of FCEL or FCEH. Treatment of an allergic disorder is alleged with a FCEL or FCEH specific polypeptide (provided the FCEH specific polypeptide is incapable of crosslinking FCEH and inducing histamine release).
EP 0269728 purports to disclose Mabs to the human lymphocyte IgE receptor.
EP 0259585 purports to disclose Mabs recognising a surface receptor for IgE (FCxcex5R) on human B lymphocytes.
WO 93/02108 purports to disclose primatised antibodies for therapeutic use.
The present inventors have surprisingly discovered that binding agents to CD23 can be of utility in the treatment or prophylaxis of various diseases and in particular in the treatment or prophylaxis of arthritis. Prior to the present invention no data has been presented which would support such a utility, despite the publication of a large number of papers in which CD23 has been discussed.
According to the present invention, there is provided a binding agent to CD23 for use in the treatment or prophylaxis of inflammatory, autoimmune or allergic diseases.
The binding agent may function by blocking the interaction between CD23 and a ligand which binds to it. In vitro assays e.g. radioimmune assays may be used to study such a blocking effect
The binding agent may be in isolated form or as part of a pharmaceutical composition. Desirably it is in sterile form. Generally speaking a binding agent which is specific for CD23 is useful in the treatment/prophylaxis disclosed.
The present inventors have demonstrated that binding agents within the scope of the present invention work in vivo in treatment or prophylaxis of inflammatory or autoimmune diseases.
This demonstration is of great significance, given the fact that many of these diseases are difficult or impossible to treat effectively, despite long standing research into their nature and causes. This is particularly the case in respect of arthritis, which often affects people in middle age and can cause them to give up work prematurely. An effective treatment of arthritis has been a long standing goal of many research groups.
Preferred binding agents include antibodies, fragments thereof or artificial constructs comprising antibodies or fragments thereof or artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such binding agents are discussed by Dougall et al in Tibtech 12, 372-379 (1994).
They include complete antibodies, F(abxe2x80x2)2 fragments, Fab fragments, Fv fragments, SCFv fragments, other fragments, CDR peptides and mimetics. These can be obtained/prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(abxe2x80x2)2 and Fab fragments (by subjecting an IgG to molecule to pepsin or papain cleavage respectively). References to xe2x80x9cantibodiesxe2x80x9d in the following description should be taken to include all of the possibilities mentioned above.
Recombinant antibodies may be used. The antibodies may be humanized; or chimaerised.
A typical preparation of a humanised antibody in which the CDRs are derived from a different species than the framework of the antibody""s variable domains is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains, and the constant domains, of the altered antibody may be derived from a human antibody. Such a humanised antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody.
Alternatively, the antibody may be a chimaeric antibody, for instance of the type described in WO 86/01533.
A chimaeric antibody according to WO 86/01533 comprises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically, the chimaeric antibody comprises both light and heavy chain variable domains. The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region, a region derived from a protein having known binding specificity, from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.
The antibody may be a human IgG such as IgG1, IgG2, IgG3, IgG4; IgM; IgA; IgE or IgD carrying rat or mouse variable regions (chimaeric) or CDRs (humanised).
Primatizing techniques may also be used, such as those disclosed in WO93/02108.
As will be appreciated by those skilled in the art, where specific binding agents are described herein, derivatives of such agents can also be used. The term xe2x80x9cderivativexe2x80x9d includes variants of the agents described, having one or more amino acid substitutions, deletions or insertions relative to said agents, whilst still having the binding activity described. Preferably these derivatives have substantial amino acid sequence identity with the binding agents specified.
The degree of amino acid sequence identity can be calculated using a program such as xe2x80x9cbestfitxe2x80x9d (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) to find the best segment of similarity between any two sequences. The alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358.
Preferably the degree of sequence identity is at least 50% and more preferably it is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred.
It will nevertheless be appreciated by the skilled person that high degrees of sequence identity are not necessarily required since various amino acids may often be substituted for other amino acids which have similar properties without substantially altering or adversely affecting certain properties of a protein. These are sometimes referred to as xe2x80x9cconservativexe2x80x9d amino acid changes. This the amino acids glycine, valine, leucine or isoleucine can often be substituted for one another (amino acids having aliphatic hydroxyl side chains). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains) and cysteine and methionine (amino acids having sulphur containing side chains). Thus the term xe2x80x9cderivativexe2x80x9d can also include a variant of an amino acid sequence comprising one or more such xe2x80x9cconservativexe2x80x9d changes relative to said sequence.
The present invention also includes fragments of the binding agents or of the present invention or of derivatives thereof which still have the binding activity described. Preferred fragments are at least ten amino acids long, but they may be longer (e.g. up to 50 or up to 100 amino acids long).
The binding agents of the present invention are believed to be useful in the treatment or prophylaxis of several human diseases including arthritis, lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowl disease, ulcerative colitis, Crohn""s disease, Sjogren""s syndrome, allergies, asthma, rhinitis, eczema, GVH, COPD, insulitis, bronchitis (particularly chronic bronchitis) or diabetes (particularly Type 1 diabetes).
They may also be useful in studying the interactions between CD23 and various ligands e.g. between CD23 and CD21, between CD23 and CD11b, between CD23 and CD11c, between CD23 and a 70 to 85 KDa endothelial cell protein (which may be a 76, 80 or 85 KDa endothelial cell protein) or between CD23 and a 115 KDa endothelial protein (which is believed to be related to the 70 to 85 KDa endothelial protein). One or more of the above interactions are believed to occur, in vivo. Antibodies or other binding agents which are capable of blocking these interactions are particularly preferred since it is believed that they may be especially suitable for reducing or alleviating cytokine mediated inflammatory effects. They may also be useful against B-cell malignancies such as chronic lymphocytic leukaemia, and hairy cell leukaemia.
Binding agents of the present invention are particularly applicable for use in the treatment or prophylaxis of rheumatoid arthritis. Without being bound by theory, the following possible explanations are put forward:
In the rheumatoid arthritis inflamed synovium, macrophages express both CD23 and the xcex22 integrins CD11b and CD11c, allowing possible homotypic interactions to take place in this tissue. In addition, diffusion of soluble CD23 molecules through the synovium and their binding to the integrin ligands is also possible. Therefore, CD23CD11b/CD11c interactions involving a positive activation loop could exist in vivo. If present in rheumatoid arthritis patients, it may explain some of the pathogenic mechanisms of disease exacerbation and chronicity, and would support the hypothesis once localised to the joints, macrophages themselves can maintain and exacerbate inflammation via a pathway involving CD23 molecules, xcex22-integrins CD11b and CD11c, as well as pro-inflammatory cytokines TNF-xcex1, IL-1xcex2 and IL-6.
An alternative mechanism of action of anti CD23 therapy could involve the blocking of an IgE immune response.
In previously published work it has been shown that in vivo treatment of rats with anti-CD23 antibody resulted in antigen-specific inhibition of IgE production, probably by blocking the CD23-CD21 interactions necessary for complete differentiation of IgE-committed B cells (Flores-Romo et al., Science 261, 1038-1041 (1993)).
The present invention also includes binding agents to CD23 which block such a response.
Structurally, the CD21 protein is composed of an extracellular domain of 15 (Moore et al, Molecular cloning of the cDNA encoding the Epstein Barr Virus C3d receptor (complement receptor type 2) of human B lymphocyte, Proc Natl Acad Sci USA 84: 9194 (1987)) or 16 (Weis et al, Structure of the human B lymphocyte receptor for C3d and the Epstein Barr Virus and relatedness to other members of the family of C3/C4 binding proteins, J Exp Med 167: 1047 (1988)) repetitive units of 60 to 75 amino acids, named short consensus repeats (SCRs), followed by a transmembrane domain (24 amino acids) and an intracytoplasmic region of 34 amino acids. Using CD21 mutants bearing deletions of extracytoplasmic SCRs (Carel et al, Structural requirements for C3d,g/Epstein Barr Virus receptor (CR2/CD21) ligand binding, internalization, and viral infection J Biol Chem 265: 12293 (1990)), the present inventors have recently found that CD23 binds to SCRs 5-8 and 1-2 on CD21. The binding of CD23 to SCRs 5-8 is a lectin-like interaction, involving carbohydrates on Asn 295 and 370. In contrast, CD23 binding to SCRs 1-2 is a protein-protein interaction (Aubry et al, CD23 interacts with a new functional extracytoplasmic domain involving N-linked oligosaccharides on CD21. J Immunol 152: 5806 (1994)). The present inventors have now tested the effect of the other ligands of CD21 (EBV, C3d,g and IFN-xcex1) on CD23 binding to CD21 and on the regulation of Ig production in the presence of IL-4. Only EBV particles and an EBV-derived peptide were able to inhibit CD23 binding to CD21. Moreover, the EBV-peptide selectively decreased IgE and IgG4 production and increased IgM production. These data indicate that CD23 binding to the EBV-binding site on CD21 selectively regulates human Ig production in the presence of IL-4.
Again without being bound by theory, it is believed that the present invention allows effective treatments to be achieved by suppressing the de novo synthesis of pro-inflammatory cytokines.
This contrasts with previous uses of antibodies simply to directly neutralise the cytokine molecules already present in inflamed tissues.
It should also be noted that there are speculative publications in the art listing large numbers of antibodies as well as large numbers of possible diseases which the antibodies are said to be useful in treating, but not providing any sound evidence or data for most of the possible combinations. One such publication is WO93/02108 which is primarily directed to the production of particular chimaeric antibodies.
The present invention is clearly distinguished from such publications by providing binding agents to particular molecules which are clearly indicated to be of utility in the treatment or prophylaxis of certain diseases in view of the data and explanations provided herein.
Binding agents of this invention are also of particular use in the treatment or prophylaxis of allergic diseases, including non-IgE mediated diseases. They may be used in the treatment and propylaxis of ulcerative colitis. They may also be used in the treatment and prophylaxis of Crohn""s disease.
The binding agents of the present invention may be used alone or in combination with immunosuppressive agents such as steroids, cyclosporin, or antibodies such as an anti-lymphocyte antibody or more preferably with a tolerance-inducing, anti-autoimmune or anti-inflammatory agent such as a CD4+T cell inhibiting agent e.g. an anti-CD4 antibody (preferably a blocking or non-depleting antibody), an anti-CD8 antibody, a TNF antagonist e.g. an anti-TNF antibody or TNF inhibitor e.g. soluble TNF receptor, or agents such as NSAIDs.
The binding agent will usually be supplied as part of a sterile, pharmaceutically acceptable composition This pharmaceutical composition may be in any suitable form, depending upon the desired method of administering it to a patient. It may be provided in unit dosage form and may be pro ed as part of a kit. Such a kit would normally (although not necessarily) include instructions for use.
Binding agent administrations are generally given parenterally, for example intravenously, intramuscularly or subcutaneously. The binding agents are generally given by injection or by infusion. For this purpose a binding agent is formulated in a pharmaceutical composition containing a pharmaceutically acceptable carrier or diluent. Any appropriate carrier or diluent may be used, for example isotonic saline solution. Stabilizers may be added such as a metal chelator to avoid copper-induced cleavage. A suitable chelator would be EDTA, DTPA or sodium citrate.
They may be given orally or nasally by means of a spray, especially for treatment of respiratory disorders.
They may be formulated as creams or ointments, especially for use in treating skin disorders.
They may be formulated as drops, or the like, for administration to the eye, for use in treating disorders such as vernal conjunctivitis.
For injectable solutions, excipients which may be used include for example, water, alcohols, polyols, glycerine, and vegetable oils.
The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants. They may also contain other therapeutically active agents.
Suitable dosages of the substance of the present invention will vary, depending upon factors such as the disease or disorder to be treated, the route of administration and the age and weight of the individual to be treated. Without being bound by any particular dosages, it is believed that for instance for parenteral administration, a daily dosage of from 0.01 to 50 mg/kg of a binding agent of the present invention (usually present as part of a pharmaceutical composition as indicated above) may be suitable for treating a typical adult. More suitably the dose might be 0.05 to 10 mg/kg, such as 0.1 to 2 mg/kg.
This dosage may be repeated as often as appropriate. Typically administration may be 1 to 7 times a weeks. If side effects develop the amount and/or frequency of the dosage can be reduced.
A typical unit dose for incorporation into a pharmaceutical composition would thus be at least 1 mg of binding agent, suitably 1 to 1000 mg.
The present invention includes within its scope an assay for determining whether or not a particular agent which binds to CD23 may be useful in the treatment of an inflammatory, autoimmune or allergic disease comprising: determining whether or not the agent is capable of blocking the interaction between CD23 and CD11b, or the interaction between CD23 and CD11c, or the interaction between CD23 and CD21, or the interaction between CD23 and a 70 to 85 KDa (such as a 76 KDa, 85 KDa or 80 KDa) or a 115 KDa protein expressed on endothelial cells.
This assay can be used for screening compounds or molecules by using cell lines expressing the appropriate molecules. Preferably CD11b is used in these assays as CD11b/CD18 and CD11c is used as CD11c/CD18. CD11b/CD18 and CD11c/CD18 can be co-expressed on cell surface.
Any appropriate assay technique can be used, e.g. protein-non protein assays (e.g. assaying the interaction of proteins with chemicals or sugars), protein-protein assays or protein-cell assays.